TWI491407B - Use of pedf-derived polypeptides for treating osteoarthritis - Google Patents
Use of pedf-derived polypeptides for treating osteoarthritis Download PDFInfo
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本發明是關於骨性關節炎之治療，且特別是關於利用來自色素上皮衍生因子(pigment epithelium-derived factor，簡稱PEDF)的合成胜肽來治療骨性關節炎。The present invention relates to the treatment of osteoarthritis, and in particular to the treatment of osteoarthritis using a synthetic peptide derived from a pigment epithelium-derived factor (PEDF).
骨性關節炎(osteoarthritis)是一種退化性關節疾病，其主要影響的是軟骨(cartilage)組織；這是一種可滑動的組織，覆蓋於骨頭末端和另一骨頭相接形成關節的部位。健康的軟骨使得骨頭可相對於另一骨頭而滑動，並可吸收物理運動時帶來的衝擊能量。在發生骨性關節炎的部位，軟骨的表層破裂並受到耗損，因而位於受損軟骨下方的骨頭會彼此摩擦，導致關節疼痛、腫脹並失去移動能力。骨性關節炎的發生率和年齡有關，最容易受影響的關節是常年承受壓力的部位，譬如膝蓋、臀部、手指與下脊椎等部位。Osteoarthritis is a degenerative joint disease that primarily affects cartilage tissue; it is a slidable tissue that covers the joint between the end of the bone and another bone to form a joint. Healthy cartilage allows the bone to slide relative to the other bone and absorbs the impact energy from physical movement. In the area where osteoarthritis occurs, the surface layer of the cartilage ruptures and is worn out, so that the bones located under the damaged cartilage rub against each other, causing joint pain, swelling and loss of mobility. The incidence of osteoarthritis is related to age. The most susceptible joints are areas that are subject to stress throughout the year, such as the knees, buttocks, fingers and lower spine.
骨性關節炎是最常見的關節炎類型。據統計，在2008年時，25歲以上的美國人當中約有2千7百萬人患有骨性關節炎。至於在全球60歲以上的人口中，據估計約有9.6%的男性與18.0%的女性具有骨性關節炎相關症狀。世界衛生組織的調查顯示，骨性關節炎的患者中，約有80%的患者行動受到限制，且約有25%的患者無法進行主要的日常活動。Osteoarthritis is the most common type of arthritis. According to statistics, in 2008, about 27 million of Americans over the age of 25 suffered from osteoarthritis. As for the population over 60 years old, it is estimated that about 9.6% of men and 18.0% of women have osteoarthritis-related symptoms. According to a World Health Organization survey, about 80% of patients with osteoarthritis have limited mobility, and about 25% of patients are unable to perform major daily activities.
目前並沒有能夠有效治癒骨性關節炎的方法或藥 物。現有的疾病管理主要著重於控制疼痛、改善僵硬與維持患者日常活動能力等方面。通常會建議患者進行物理治療，此種療法有助於強化患者的肌肉與骨骼、增加肌肉撓曲性且可減低疼痛。用於骨性關節炎的藥物主要是止痛藥，譬如口服或局部塗抹的止痛劑，這些藥物雖然能夠舒緩不適，但無法治療發炎。口服或注射用的皮質類固醇(corticosteroids)可控制發炎，但不適合長期服用。非固醇類的抗發炎藥物(nonsteroidal anti-inflammatory drugs，簡稱NSAIDs)可用以減緩疼痛、腫脹與發炎等情形；然而此類藥物會導致胃部不適與潰瘍，且可能增加某些患者心臟病發作的機率。對於嚴重受損的關節，可考慮外科手術，如關節鏡手術(arthroscopic procedures)乃至於全關節造型術(total joint arthroplasty)。There are currently no methods or medicines that can effectively cure osteoarthritis. Things. Existing disease management focuses on controlling pain, improving stiffness, and maintaining the patient's ability to perform daily activities. Patients are usually advised to undergo physical therapy, which helps strengthen the patient's muscles and bones, increase muscle flexibility and reduce pain. The drugs used for osteoarthritis are mainly painkillers, such as oral or topical analgesics. Although these drugs can relieve discomfort, they cannot treat inflammation. Oral or injectable corticosteroids control inflammation, but are not suitable for long-term use. Nonsteroidal anti-inflammatory drugs (NSAIDs) can be used to relieve pain, swelling and inflammation; however, such drugs can cause stomach upsets and ulcers and may increase heart attacks in some patients. The chance. For severely damaged joints, surgery can be considered, such as arthroscopic procedures or total joint arthroplasty.
有鑑於上述問題，相關領域亟需提出一種能夠有效治療骨性關節炎的藥物與方法。In view of the above problems, there is an urgent need for a drug and method for effectively treating osteoarthritis in related fields.
發明內容旨在提供本揭示內容的簡化摘要，以使閱讀者對本揭示內容具備基本的理解。此發明內容並非本揭示內容的完整概述，且其用意並非在指出本發明實施例的重要/關鍵元件或界定本發明的範圍。發明內容旨在提供本揭示內容的簡化摘要，以使閱讀者對本揭示內容具備基本的理解。此發明內容並非本揭示內容的完整概述，且其用意並非在指出本發明實施例的重要/關鍵元件 或界定本發明的範圍。SUMMARY OF THE INVENTION The Summary of the Disclosure is intended to provide a basic understanding of the present disclosure. This Summary is not an extensive overview of the disclosure, and is not intended to be an SUMMARY OF THE INVENTION The Summary of the Disclosure is intended to provide a basic understanding of the present disclosure. This Summary is not an extensive overview of the disclosure, and is not intended to identify important/critical elements of the embodiments of the present invention. Or define the scope of the invention.
本發明至少部分是基於發現PEDF衍生合成胜肽(下稱合成胜肽或PEDF胜肽)能夠刺激軟骨細胞(chondrocytes)擴增、引發軟骨再生並促進間葉幹細胞的軟骨細胞生成(chondrogenesis)；因而這些PEDF胜肽能夠有效治療一個體之骨性關節炎。由此可知，此處提出的PEDF衍生合成胜肽能夠作為用以治療骨性關節炎的活性化合物。The present invention is based, at least in part, on the discovery that a PEDF-derived synthetic peptide (hereinafter referred to as a synthetic peptide or a PEDF peptide) is capable of stimulating the expansion of chondrocytes, triggering cartilage regeneration, and promoting chondrogenesis of mesenchymal stem cells; These PEDF peptides are effective in treating a body of osteoarthritis. From this, it can be seen that the PEDF-derived synthetic peptide proposed herein can be used as an active compound for treating osteoarthritis.
有鑑於此，本發明的一態樣是關於一種PEDF衍生合成胜肽的用途，此PEDF衍生合成胜肽可用以製備治療一個體之骨性關節炎的醫藥品。In view of this, one aspect of the present invention relates to the use of a PEDF-derived synthetic peptide which can be used to prepare a medicament for treating a body of osteoarthritis.
根據本說明書多個實施例，所述的合成胜肽長度為20-39個胺基酸殘基，且其序列至少80%與序列編號：1相同。此外，上述胺基酸序列包含至少20個連續胺基酸殘基，其序列至少90%與序列編號：1的胺基酸殘基11-30相同，而使得此合成胜肽可用於治療一個體的骨性關節炎。According to various embodiments of the present specification, the synthetic peptide is 20-39 amino acid residues in length, and the sequence thereof is at least 80% identical to the sequence number: 1. Furthermore, the above amino acid sequence comprises at least 20 contiguous amino acid residues, the sequence of which is at least 90% identical to the amino acid residues 11-30 of SEQ ID NO: 1, such that the synthetic peptide can be used to treat a body. Osteoarthritis.
在可任選的實施例中，上述合成胜肽中有至少四個連續的胺基酸，其序列與序列編號：1的胺基酸殘基11-14相同。此種合成胜肽的非限制性例示包括胺基酸序列如序列編號：1(39-mer)、序列編號：2(34-mer)、序列編號：3(29-mer)、序列編號：5(24-mer)、序列編號：6(20-mer)、序列編號：8(MO 29-mer)與序列編號：9(MO 20-mer)所示者。根據本發明某些實施例，上述合成胜肽的胺基酸序列為序列編號：3(29-mer)、序列編號：5(24-mer)或 序列編號：6(20-mer)任一者所示。In an optional embodiment, the above synthetic peptide has at least four consecutive amino acids having the same sequence as the amino acid residues 11-14 of SEQ ID NO: 1. Non-limiting examples of such synthetic peptides include amino acid sequences such as SEQ ID NO: 1 (39-mer), SEQ ID NO: 2 (34-mer), SEQ ID NO: 3 (29-mer), SEQ ID NO: 5 (24-mer), SEQ ID NO: 6 (20-mer), SEQ ID NO: 8 (MO 29-mer) and SEQ ID NO: 9 (MO 20-mer). According to some embodiments of the invention, the amino acid sequence of the above synthetic peptide is SEQ ID NO: 3 (29-mer), SEQ ID NO: 5 (24-mer) or Serial number: 6 (20-mer) is shown as either.
根據本發明多種實施例，所述的個體可以是任何哺乳類動物，包括人類。According to various embodiments of the invention, the individual may be any mammal, including a human.
在本發明另一態樣中，提出一種用以治療一個體之骨性關節炎的藥學組合物。所述的個體可以是任何哺乳類動物，包括人類。In another aspect of the invention, a pharmaceutical composition for treating osteoarthritis in a body is provided. The individual can be any mammal, including a human.
根據本發明一實施方式，上述組合物包含任一根據本發明上述態樣/實施例的合成胜肽，且此合成胜肽的含量足以治療該個體的骨性關節炎。此組合物亦包含可用以攜帶該合成胜肽的一種藥學上可接受賦型劑。According to an embodiment of the present invention, the above composition comprises any of the synthetic peptides according to the above aspect/embodiment of the present invention, and the synthetic peptide is present in an amount sufficient to treat osteoarthritis in the individual. The composition also includes a pharmaceutically acceptable excipient that can be used to carry the synthetic peptide.
於可任選的實施例中，所述藥學組合物更包含一糖胺聚多醣(glycosaminoglycan)，譬如玻尿酸(hyaluronic acid)或玻尿酸鈉(sodium hyaluronate)。In an optional embodiment, the pharmaceutical composition further comprises a glycosaminoglycan such as hyaluronic acid or sodium hyaluronate.
根據本發明多種實施例，可將所述藥學組合物製成可注射的劑型。According to various embodiments of the invention, the pharmaceutical composition can be formulated as an injectable dosage form.
本發明的又一態樣是一種用以治療一個體之骨性關節炎的方法。所述的個體可以是任何哺乳類動物，包括人類。Yet another aspect of the invention is a method of treating osteoarthritis in a body. The individual can be any mammal, including a human.
於一實施例中，上述方法包含對該個體投予根據本發明上述態樣/實施例的合成胜肽，以使得此合成胜肽到達該個體的滑液囊(synovial cavity)內，進而治療骨性關節炎。In one embodiment, the method comprises administering to the individual a synthetic peptide according to the above aspect/embodiment of the invention such that the synthetic peptide reaches the synovial cavity of the individual, thereby treating the bone Arthritis.
於可任選的本發明實施例中，可將合成胜肽調製為根據上述態樣實施例所述的組合物。In an optional embodiment of the invention, the synthetic peptide can be formulated into a composition according to the above-described embodiment.
於某些實施例中，可利用關節內注射(intra-articular injection)的方式將所述合成胜肽或藥學組合物注射至該 個體的滑液囊。In certain embodiments, the synthetic peptide or pharmaceutical composition can be injected into the body using intra-articular injection. Individual synovial sac.
在參閱下文實施方式後，本發明所屬技術領域中具有通常知識者當可輕易瞭解本發明之基本精神及其他發明目的，以及本發明所採用之技術手段與實施態樣。The basic spirit and other objects of the present invention, as well as the technical means and implementations of the present invention, will be readily apparent to those skilled in the art of the invention.
為了使本揭示內容的敘述更加詳盡與完備，下文針對了本發明的實施態樣與具體實施例提出了說明性的描述；但這並非實施或運用本發明具體實施例的唯一形式。實施方式中涵蓋了多個具體實施例的特徵以及用以建構與操作這些具體實施例的方法步驟與其順序。然而，亦可利用其他具體實施例來達成相同或均等的功能與步驟順序。The description of the embodiments of the present invention is intended to be illustrative and not restrictive. The features of various specific embodiments, as well as the method steps and sequences thereof, are constructed and manipulated in the embodiments. However, other specific embodiments may be utilized to achieve the same or equivalent function and sequence of steps.
除非本說明書另有定義，此處所用的科學與技術詞彙之含義與本發明所屬技術領域中具有通常知識者所理解與慣用的意義相同。此外，在不和上下文衝突的情形下，本說明書所用的單數名詞涵蓋該名詞的複數型；而所用的複數名詞時亦涵蓋該名詞的單數型。The scientific and technical terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention pertains, unless otherwise defined herein. In addition, the singular noun used in this specification covers the plural of the noun in the case of no conflict with the context; the plural noun of the noun is also included in the plural noun used.
雖然用以界定本發明較廣範圍的數值範圍與參數界是約略的數值，此處已盡可能精確地呈現具體實施例中的相關數值。然而，任何數值本質上不可避免地含有因個別測試方法所致的標準偏差。在此處，「約」通常係指實際數值在一特定數值或範圍的正負10%、5%、1%或0.5%之內。或者是，「約」一詞代表實際數值落在平均值的可接受標準 誤差之內，視本發明所屬技術領域中具有通常知識者的考量而定。除了實驗例之外，或除非另有明確的說明，當可理解此處所用的所有範圍、數量、數值與百分比(例如用以描述材料用量、時間長短、溫度、操作條件、數量比例及其他相似者)均經過「約」的修飾。因此，除非另有相反的說明，本說明書與附隨申請專利範圍所揭示的數值參數皆為約略的數值，且可視需求而更動。至少應將這些數值參數理解為所指出的有效位數與套用一般進位法所得到的數值。Although numerical ranges and parameter boundaries are used to define a broad range of values for the present invention, the relevant values in the specific embodiments are presented as precisely as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range. Or, the word "about" represents the acceptable standard for the actual value to fall on the average. Within the error, it will be considered by those of ordinary skill in the art to which the invention pertains. Except for the experimental examples, or unless otherwise explicitly stated, all ranges, quantities, values, and percentages used herein are understood (eg, to describe the amount of material used, the length of time, the temperature, the operating conditions, the quantity ratio, and the like. Are all modified by "about". Therefore, unless otherwise indicated to the contrary, the numerical parameters disclosed in the specification and the appended claims are intended to be At a minimum, these numerical parameters should be understood as the number of significant digits indicated and the values obtained by applying the general carry method.
在此處「胜肽」(peptide)一詞係指胺基酸殘基所組成的高分子。「合成胜肽」(synthetic peptide)一詞則代表此胜肽並未包含存在於自然界的完整蛋白質分子。此種胜肽之所以是「合成的」，表示其乃是由人類利用技術手段所得，譬如化學合成、重組遺傳技術或將整個抗原切段。於本說明書中，任何胺基酸殘基於一胜肽中的位置係由該胜肽的N端起算。The term "peptide" as used herein refers to a polymer composed of amino acid residues. The word "synthetic peptide" means that the peptide does not contain intact protein molecules found in nature. The reason why such a peptide is "synthetic" is that it is obtained by humans using technical means such as chemical synthesis, recombinant genetic techniques or segmentation of the entire antigen. In the present specification, any amino acid residue based on the position in a peptide is counted from the N-terminus of the peptide.
在此處「增殖」的各種詞性(如proliferate或proliferation)係指族群中的細胞數目透過細胞***而增加之情形。The various parts of the word "proliferation" or "proliferation" herein refer to the situation in which the number of cells in a population increases through cell division.
此處針對合成胜肽序列所述的「胺基酸序列相似度百分比」(Percent% amino acid sequence identity)係指該候選合成胜肽之胺基酸殘基與一參考多胜肽之胺基酸殘基完全相同的百分比。於進行上述比對時，可將該候選合成胜肽與該參考多胜肽並排，並於必要時引入間隙，以使二序
列形成最高的序列相似度，且在計算相似度時，並未將保守性置換之胺基酸殘基納入考量。相關領域已有多種方法可供進行上述並排，譬如可公開取得的軟體如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)等。本發明所屬技術領域中具有通常知識者在進行並排時，可選擇適當的參數與計算方式，以得到最佳的排列方式。在本說明書中，二胺基酸序列間的序列比較是採用美國國家生物科技資訊中心(Nation Center for Biotechnology Information，簡稱NCBI)所提供的蛋白質-蛋白質BLAST分析軟體Blastp來進行。一候選胺基酸序列A相較於一參考胺基酸序列B的胺基酸序列相似度(在本說明書中亦稱之為序列A與序列B具有特定百分比(%)的胺基酸序列相似度)的計算方式如下：
於本說明書中，「治療」一詞的各種詞性與時態(如treat、treating或treatment)係指預防性(如，預防用藥)、療癒性或緩和性的處置，藉以達到所欲的藥學和/或生理學效果。在較佳的情形中，上述的效果是指能夠部分或完全地治癒個體的骨性關節炎。此外，「治療」一詞可用以指稱將此處提出的PEDF衍生合成胜肽或藥學組合物投予或施用於一個體，此個體可能有一醫療疾患、症狀、疾病或與 疾患相關的異常或易於罹患一疾患，以期能部分地或完全地緩和、改善、減輕一特定異常和/或病症的一或多種症狀或特徵，或延緩其發生、阻礙其進展、減輕其嚴重性和/或減低發生率。亦可對並未表出現疾病、異常和/或病症之徵兆的個體和/或呈現早期徵兆的個體進行治療，以期降低發展出與該疾病、異常和/或病症相關之病理變化的風險。於此處，上述症狀、疾病、異常或病症係指骨性關節炎。在本說明書中，當一或更多種症狀或臨床指標減少時，通常認為該治療是「有效」的。In this specification, the various parts of speech and tense (such as treat, treating or treatment) refer to the prophylactic (eg, prophylactic), healing or palliative treatment to achieve the desired pharmacy. And / or physiological effects. In the preferred case, the above effect means that the osteoarthritis of the individual can be partially or completely cured. Furthermore, the term "treatment" may be used to refer to the administration or administration of a PEDF-derived synthetic peptide or pharmaceutical composition presented herein to a body which may have a medical condition, symptom, disease or A disorder associated with a condition or a condition that is prone to suffer, in order to partially or completely alleviate, ameliorate, alleviate, or delay the onset, hinder its progression, and reduce the severity of a particular abnormality and/or condition. And / or reduce the incidence. Individuals who are not indicative of signs of disease, abnormality, and/or condition and/or individuals presenting early signs may also be treated with a view to reducing the risk of developing pathological changes associated with the disease, disorder, and/or condition. Here, the above symptoms, diseases, abnormalities or conditions refer to osteoarthritis. In the present specification, when one or more symptoms or clinical indicators are reduced, the treatment is generally considered to be "effective."
在此處，「有效量」(effective amount)一詞係指一成分的用量足以招致所欲的反應或效果。具體的有效量取決於多種因素，諸如欲處理的特定狀況、個體的生理條件(如，個體體重、年齡或性別)、接受處置的哺乳動物或動物的類型、處置持續時間、目前所受其他治療(如果有的話)的本質以及所用的具體配方和化合物或其衍生物的結構。有效量亦指於此用量下，該化合物或組合物的毒性或負面效果不及於其所帶來的正面效果。As used herein, the term "effective amount" means an amount of a component sufficient to induce a desired response or effect. The specific effective amount depends on various factors such as the particular condition to be treated, the individual's physiological condition (eg, individual body weight, age or sex), the type of mammal or animal being treated, the duration of treatment, and other current treatments The nature of (if any) and the specific formulation used and the structure of the compound or derivative thereof. An effective amount also means that the toxicity or negative effect of the compound or composition is less than the positive effect brought about by this amount.
「施用」或「投予」(application or administration)等詞在此係指將一或多劑有效量的所述PEDF衍生合成胜肽或組合物提供給個體，以治療骨性關節炎。根據本發明實施方式，較佳的遞送方式為關節內注射(intra-articular injection)。舉例來說，可將所述之PEDF衍生合成胜肽或藥學組合物透過關節內注射到個體關節的滑液囊內，以促進軟骨再生，進而治療骨性關節炎。The word "application" or "administration" as used herein refers to the administration of one or more effective amounts of the PEDF-derived synthetic peptide or composition to an individual for the treatment of osteoarthritis. According to an embodiment of the invention, the preferred mode of delivery is intra-articular injection. For example, the PEDF-derived synthetic peptide or pharmaceutical composition can be injected intra-articularly into the synovial sac of an individual's joint to promote cartilage regeneration, thereby treating osteoarthritis.
此處「賦型劑」(excipient)一詞係指可作為所述PEDF衍生合成胜肽的媒劑/載體之任何惰性物質(包括粉末或溶液)。賦型劑通常是安全無毒性的物質，且廣義上包括製藥產業中可用以製備組合物的任何物質，如填充劑、稀釋劑、凝結劑、黏合劑、潤滑劑、助流劑、安定劑、著色劑、浸潤劑等。The term "excipient" as used herein refers to any inert substance (including powder or solution) which can be used as a vehicle/carrier for the PEDF-derived synthetic peptide. Excipients are generally safe, non-toxic materials, and broadly include any material that can be used in the pharmaceutical industry to prepare compositions, such as fillers, diluents, coagulants, binders, lubricants, glidants, stabilizers, Colorants, sizing agents, etc.
在本說明書中，「藥學上可接受」(pharmaceutically acceptable)的成分係指其適用於人類和/或動物，且在合理的效益/風險比之下不會產生不當的副作用(如毒性、刺激與過敏反應)。此外，每一種賦型劑必須和藥學配方中的其他成分相容，才是「可接受」的成分。In the present specification, "pharmaceutically acceptable" means that it is suitable for use in humans and/or animals and does not cause undue side effects (such as toxicity, irritation, and toxicity) at a reasonable benefit/risk ratio. Allergic reaction). In addition, each excipient must be compatible with the other ingredients in the pharmaceutical formulation to be an "acceptable" ingredient.
「個體」(subject)一詞在此係指可接受本發明之合成胜肽、組合物和/或方法來進行治療的哺乳類動物(包括人類)。除非另有指明，「個體」一般包含雄性與雌性。The term "subject" as used herein refers to a mammal (including a human) that can receive the synthetic peptides, compositions and/or methods of the invention for treatment. Unless otherwise indicated, "individual" generally includes males and females.
色素上皮衍生因子(Pigment epithelium-derived factor，簡稱PEDF)是一種多功能的分泌性蛋白質，其具有抗血管新生(anti-angiogenic)、抗腫瘤生成(anti-tumorigenic)與神經滋養(neurotrophic)等功能。人類PEDF蛋白質(序列編號：11)是一種大小約50 kDa的分泌性蛋白質，具有418個胺基酸。已知PEDF的34-mer片段(相當於PEDF之第44-77號殘基)與44-mer片段(相當於PEDF之第78-121號殘基；序列編號：10)分別具有抗血管新生與神經滋養性質。Pigment epithelium-derived factor (PEDF) is a multifunctional secreted protein with anti-angiogenic, anti-tumorigenic and neurotrophic functions. . The human PEDF protein (SEQ ID NO: 11) is a secreted protein of approximately 50 kDa with 418 amino acids. It is known that the 34-mer fragment of PEDF (corresponding to residues 44-77 of PEDF) and the 44-mer fragment (corresponding to residues 78-121 of PEDF; SEQ ID NO: 10) have antiangiogenic effects, respectively. Nerve nourishing properties.
本說明書至少部分是基於發現來自44-mer PEDF的合 成胜肽能夠透過多種機制來促進軟骨再生。本說明書提供的實驗例證實，所述的合成胜肽能夠促進軟骨細胞擴增且可提高間葉幹細胞的軟骨細胞生成。本發明的另一創新特徵在於此處所提出的合成胜肽最長只有20至39個胺基酸殘基，比起先前技術所述的全長PEDF短了許多；因此，本發明克服了傳統蛋白質在臨床使用上經常面臨的困境，諸如製造成本高昂、生體可用率(bioavailability)低落與藥物動力學(pharmacokinetics)表現不佳等。因此，這些合成胜肽可用以治療骨性關節炎。This specification is based, at least in part, on the discovery of combinations from 44-mer PEDF. Chengsheng peptide can promote cartilage regeneration through a variety of mechanisms. The experiments provided in the present specification demonstrate that the synthetic peptide can promote chondrocyte expansion and increase chondrocyte production of mesenchymal stem cells. Another innovative feature of the present invention is that the synthetic peptides proposed herein have a maximum length of only 20 to 39 amino acid residues, which is much shorter than the full length PEDF described in the prior art; therefore, the present invention overcomes the traditional protein in clinical practice. Difficulties often encountered in use, such as high manufacturing costs, low bioavailability, and poor pharmacokinetics. Therefore, these synthetic peptides can be used to treat osteoarthritis.
有鑑於此，本發明的一態樣是關於一種PEDF衍生合成胜肽用於治療一個體之骨性關節炎的用途。本發明的另一態樣是關於上述PEDF衍生合成胜肽的另一種用途，其可用於製備用以治療一個體之骨性關節炎的藥學組合物。除了上述用途之外，本發明的一態樣亦提出了一種治療一對象之骨性關節炎的方法。下文記載了適用於以上各態樣的多種實施例。In view of this, one aspect of the present invention relates to the use of a PEDF-derived synthetic peptide for the treatment of osteoarthritis in a body. Another aspect of the present invention pertains to another use of the above PEDF-derived synthetic peptide which can be used in the preparation of a pharmaceutical composition for treating osteoarthritis in a body. In addition to the above uses, an aspect of the present invention also provides a method of treating osteoarthritis in a subject. Various embodiments suitable for the above aspects are described below.
根據本說明書多個實施例，此合成胜肽有20-39個胺基酸殘基，且其胺基酸序列與序列編號：1(LSVATALSALSLGAEQRTESIIHRALYYDLISSPDIHGT)有至少80%的胺基酸序列相似度。舉例來說，此合成胜肽與序列編號：1的胺基酸序列相似度可為約80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99或100%。另外，此合成胜肽包含至少20個連續的胺基酸殘基，其與序列編號：1第11-30個殘基 有至少90%的胺基酸序列相似度。具體來說，這20個連續胺基酸殘基與序列編號：1第11-30個殘基的胺基酸序列相似度可為約90、91、92、93、94、95、96、97、98、99或100%。According to various embodiments of the present specification, the synthetic peptide has 20 to 39 amino acid residues, and the amino acid sequence thereof has at least 80% amino acid sequence similarity to SEQ ID NO: 1 (LSVATALSALSLGAEQRTESIIHRALYYDLISSPDIHGT). For example, the amino acid sequence sequence of the synthetic peptide and SEQ ID NO: 1 can be about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93. , 94, 95, 96, 97, 98, 99 or 100%. In addition, the synthetic peptide comprises at least 20 consecutive amino acid residues which are SEQ ID NO: 1 11-30 residues There is at least 90% amino acid sequence similarity. Specifically, the 20 consecutive amino acid residues may have a similarity to the amino acid sequence of the 11th to 30th residues of SEQ ID NO: 1 of about 90, 91, 92, 93, 94, 95, 96, 97. , 98, 99 or 100%.
在本發明一實施例中，所述的合成胜肽有39個胺基酸殘基，其序列即如序列編號：1所示。在下文實驗例中，亦將此合成胜肽稱為39-mer。此種39-mer胜肽是來自人類PEDF的已知44-mer片段(PEDF第78-121號殘基)的一種較短的變形。In one embodiment of the invention, the synthetic peptide has 39 amino acid residues, the sequence of which is as shown in SEQ ID NO: 1. In the experimental examples below, this synthetic peptide was also referred to as a 39-mer. Such a 39-mer peptide is a shorter variant of the known 44-mer fragment from human PEDF (PEDF residues 78-121).
本案發明人先前所做的實驗(如，審查中的美國專利申請案第13/428996號，在此將該先申請案的內容一併納入為本說明書的一部分)與本說明書所提供的實驗例顯示還有其他數種來自此39-mer的短PEDF合成胜肽能夠用以治療骨性關節炎。The experiments previously performed by the inventors of the present invention (e.g., U.S. Patent Application Serial No. 13/428,996, the entire disclosure of which is incorporated herein by reference in its entirety) There are several other short PEDF synthetic peptides from this 39-mer that can be used to treat osteoarthritis.
譬如，由下文與先申請案的實驗例可知，如序列編號：2(ALSALSLGAEQRTESIIHRALYYDLISSPDIHGT)所示的34-mer合成胜肽能夠有效地治療骨性關節炎。此34-mer合成胜肽相當於人類PEDF的第88-121號胺基酸殘基；根據上文所述的胺基酸序列相似度的計算方法，此34-mer與39-mer的胺基酸序列相似度為100%，且34-mer的第6-25號胺基酸殘基與39-mer的第11-30號胺基酸殘基的胺基酸序列相似度也是100%。For example, from the experimental examples of the following and the prior application, the 34-mer synthetic peptide as shown in SEQ ID NO: 2 (ALSALSLGAEQRTESIIHRALYYDLISSPDIHGT) is effective for the treatment of osteoarthritis. The 34-mer synthetic peptide corresponds to amino acid residues 88-121 of human PEDF; the 34-mer and 39-mer amine groups are calculated according to the amino acid sequence similarity calculation method described above. The acid sequence similarity is 100%, and the amino acid sequence residues of amino acids 6 to 25 of the 34-mer and the amino acid residues of the 39-mer amino acid residues are also 100%.
此外，下文多個實驗例證明如序列編號：3(SLGAEQRTESIIHRALYYDLISSPDIHGT)所示的29-mer 合成胜肽能夠有效治療個體的骨性關節炎。此29-mer合成胜肽相當於人類PEDF的第93-121號胺基酸殘基；其與39-mer的胺基酸序列相似度為100%，且29-mer的第1-20號胺基酸殘基與39-mer的第11-30號胺基酸殘基的胺基酸序列相似度亦為100%。In addition, the following experimental examples illustrate 29-mer as shown in SEQ ID NO: 3 (SLGAEQRTESIIHRALYYDLISSPDIHGT) Synthetic peptides are effective in treating osteoarthritis in individuals. This 29-mer synthetic peptide corresponds to amino acid residues 93-121 of human PEDF; its similarity to the amino acid sequence of 39-mer is 100%, and the amine of No. 1-20 of 29-mer The amino acid sequence has a similarity to the amino acid sequence of the amino acid residues 11-30 of the 39-mer.
下文某些實驗例證實，一種24-mer合成胜肽亦可有效治療個體的骨性關節炎。此24-mer的序列如序列編號：5(SLGAEQRTESIIHRALYYDLISSP)所示，且相當於人類PEDF第93-116號胺基酸殘基。此外，24-mer序列與39-mer的第11-30號胺基酸殘基完全相同(胺基酸序列相似度：100%)且其相較於39-mer的胺基酸序列相似度同樣也是100%。As demonstrated in some of the experiments below, a 24-mer synthetic peptide is also effective in treating osteoarthritis in an individual. The sequence of this 24-mer is shown in SEQ ID NO: 5 (SLGAEQRTESIIHRALYYDLISSP) and corresponds to amino acid residues of human PEDF No. 93-116. In addition, the 24-mer sequence is identical to the amino acid residues 11-30 of the 39-mer (amino acid sequence similarity: 100%) and is similar to the amino acid sequence similarity of the 39-mer. It is also 100%.
本說明書某些實驗例顯示，如序列編號：6(SLGAEQRTESIIHRALYYDL)所示的20-mer亦可有效治療個體的骨性關節炎。此20-mer的序列相當於人類PEDF第93-112號胺基酸殘基，其與39-mer的第11-30號胺基酸殘基完全相同(胺基酸序列相似度：100%)且其相較於39-mer的胺基酸序列相似度同樣也是100%。Certain experimental examples of the present specification show that the 20-mer as shown in SEQ ID NO: 6 (SLGAEQRTESIIHRALYYDL) is also effective for treating osteoarthritis in an individual. The sequence of this 20-mer corresponds to the amino acid residue of human PEDF No. 93-112, which is identical to the amino acid residues 11-30 of the 39-mer (amino acid sequence similarity: 100%) And its similarity to the amino acid sequence of the 39-mer is also 100%.
本案與先申請案所揭載的實驗例亦顯示，另外有兩種衍生自小鼠PEDF的短、合成胜肽同樣也能夠有效地治療骨性關節炎。第一種衍生自小鼠的合成胜肽在本說明書中稱為Mo 29-mer，其序列如序列編號：8(SLGAEHRTESVIHRALYYDLITNPDIHST)所示，此序列與39-mer的胺基酸序列相似度為83%，且其前20個胺基 酸殘基與39-mer第11-30個殘基的胺基酸序列相似度為90%。另一種衍生自小鼠PEDF的合成胜肽稱為Mo 20-mer，其序列如序列編號：9(SLGAEHRTESVIHRALYYDL)所示。Mo 20-mer與39-mer或39-mer的第11-30號胺基酸殘基的胺基酸序列相似度皆為90%。The experimental examples disclosed in this case and the prior application also show that two other short peptides derived from mouse PEDF can also effectively treat osteoarthritis. The first synthetic peptide derived from mouse is referred to in this specification as Mo 29-mer, the sequence of which is shown in SEQ ID NO: 8 (SLGAEHRTESVIHRALYYDLITNPDIHST), which has a similarity to the 39-mer amino acid sequence of 83. %, and its first 20 amine groups The acid residue has a similarity to the amino acid sequence of the 11-30 residues of the 39-mer of 90%. Another synthetic peptide derived from mouse PEDF is called Mo 20-mer and its sequence is shown as SEQ ID NO: 9 (SLGAEHRTESVIHRALYYDL). The amino acid sequence residues of the Mo 20-mer and the 39-mer or 39-mer amino acid residues are all 90% similar.
在可任選的實施例中，所述的PEDF衍生合成胜肽中有至少四個連續的胺基酸，其序列與序列編號：1的胺基酸殘基11-14相同。本案發明人所進行的實驗結果顯示，序列編號：1所示序列的第11-14號胺基酸殘基(即，SLGA)對於維持短PEDF合成胜肽之生理功能扮演的重要的角色。舉例來說，下文提出的多個實驗例顯示，不具有此SLGA片段的18-mer合成胜肽(EQRTESIIHRALYYDLIS；序列編號：7)無法保護個體對抗骨性關節炎。此外，由本案與先申請案所載的實驗例可以推知，不含SLGA序列的25-mer合成胜肽(EQRTESIIHRALYYDLISSPDIHGT；序列編號：4)也無法有效治療骨性關節炎。In an optional embodiment, the PEDF-derived synthetic peptide has at least four consecutive amino acids in the same sequence as the amino acid residues 11-14 of SEQ ID NO: 1. The experimental results conducted by the inventors of the present invention show that the amino acid residues No. 11-14 of the sequence of SEQ ID NO: 1 (i.e., SLGA) play an important role in maintaining the physiological function of the short PEDF synthetic peptide. For example, the various experimental examples presented below show that an 18-mer synthetic peptide (EQRTESIIHRALYYDLIS; SEQ ID NO: 7) without this SLGA fragment does not protect individuals against osteoarthritis. Furthermore, it can be inferred from the experimental examples contained in the present application and the prior application that the 25-mer synthetic peptide (EQRTESIIHRALYYDLISSPDIHGT; SEQ ID NO: 4) which does not contain the SLGA sequence is also ineffective in the treatment of osteoarthritis.
可利用任何習用的技術來合成此處所述的合成胜肽，譬如t-BOC或FMOC來保護α-氨基(alpha-amino groups)。這兩種方法都採用了逐步合成法，其係由該胜肽的C端開始，每次加上一個胺基酸。亦可利用其他已知的固態胜肽合成(solid phase peptide synthesis，簡稱SPPS)法來合成此處所述的合成胜肽。The synthetic peptides described herein, such as t-BOC or FMOC, can be synthesized using any conventional technique to protect alpha-amino groups. Both methods employ a stepwise synthesis starting with the C-terminus of the peptide, each time adding an amino acid. Other known solid phase peptide synthesis (SPPS) methods can also be utilized to synthesize the synthetic peptides described herein.
本發明之範圍亦涵蓋了其他相較於39-mer具有保守性 置換(conservative variation)的合成胜肽。在此處，「保守性置換」一詞係指利用另一種在生物學上相似的殘基來取代某一殘基。保守性置換的例示包括親水性殘基(如異白胺酸、纈胺酸、白胺酸與甲硫胺酸)彼此間的置換，或相近極性殘基(如精胺酸與離胺酸；或麩胺酸與天冬胺酸)彼此間的置換，以及其他類似的置換模式。「保守性置換」在此亦指利用一具有取代基的胺基酸來取代一不具有取代基的原始胺基酸，只要可和此具有取代基之胺基酸反應的抗體亦可和原始胺基酸進行免疫反應即可。The scope of the invention also covers other aspects that are conservative compared to 39-mer Synthetic peptides of conservative variation. Here, the term "conservative substitution" refers to the replacement of a residue with another biologically similar residue. Illustrative of conservative substitutions include substitutions of hydrophilic residues (such as isoleucine, valine, leucine, and methionine) with each other, or similar polar residues (such as arginine and lysine; Or glutamic acid and aspartate) replacement with each other, and other similar modes of substitution. "Conservative substitution" as used herein also refers to the use of a substituted amino acid to replace an unsubstituted amino acid, as long as the antibody reactive with the substituted amino acid can also react with the original amine. The base acid can be used for an immune reaction.
此處所述的PEDF多胜肽片段可用於治療哺乳類動物的骨性關節炎。所述的哺乳類動物包括但不限於人類、人類以外的靈長類、鼠科動物等。在較佳的實施例中，所述的對象為人類。The PEDF polypeptide fragments described herein are useful for the treatment of osteoarthritis in mammals. The mammals include, but are not limited to, humans, primates other than humans, murines, and the like. In a preferred embodiment, the object is a human.
亦可將上述實施方式所述的各種合成胜肽調製成一藥學組合物，以治療個體的骨性關節炎；此種藥學組合物即屬於本發明另一態樣之範圍。The various synthetic peptides described in the above embodiments may also be formulated into a pharmaceutical composition for treating osteoarthritis in an individual; such pharmaceutical compositions are within the scope of another aspect of the invention.
根據本發明一實施方式，上述藥學組合物包含任一根據本發明上述態樣/實施例的合成胜肽，且此合成胜肽的含量足以治療該個體的骨性關節炎。此藥學組合物亦包含可用以攜帶該合成胜肽的一藥學上可接受賦型劑。According to an embodiment of the present invention, the pharmaceutical composition comprises any of the synthetic peptides according to the above aspect/embodiment of the present invention, and the synthetic peptide is present in an amount sufficient to treat osteoarthritis in the individual. The pharmaceutical composition also includes a pharmaceutically acceptable excipient that can be used to carry the synthetic peptide.
根據本發明的可任選實施例，此藥學組合物中所述合成胜肽的含量為約1-1,000 μM；較佳為約10-500 μM；更佳為25-250 μM。舉例來說，上述合成胜肽的含量可為約1、2、3、4、5、6、7、8、9、10、15、20、25、30、35、 40、45、50、55、60、65、70、75、80、85、90、95、100、150、200、250、300、350、400、450、500、550、600、650、700、750、800、850、900、950或1,000 μM。具體而言，下文實驗例中針對約310克重的大鼠所用的劑量濃度為約25 μM。本發明所屬技術領域具有通常知識者可基於本說明書提出的小鼠給藥劑量，計算出人體等效劑量(human equivalent dose，簡稱HEQ)；譬如可依據美國食品暨衛生管理局提出的準則(標題為”Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers”)來計算。According to an optional embodiment of the present invention, the synthetic peptide is present in the pharmaceutical composition in an amount of from about 1 to 1,000 μM; preferably from about 10 to 500 μM; more preferably from 25 to 250 μM. For example, the content of the above synthetic peptide may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1,000 μM. Specifically, the dose concentration for the approximately 310 gram rat was used in the experimental examples below to be about 25 μM. The person skilled in the art can calculate the human equivalent dose (HEQ) based on the dose of the mouse administered in the present specification; for example, according to the guidelines proposed by the US Food and Health Administration (title) It is calculated as "Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers".
可根據既有的藥學程序來製備上述藥學組合物，譬如Remington’s Pharmaceutical Sciences(17th edition,ed.Alfonoso R.Gennaro,Mack Publishing Company,Easton,Pa(1985).)一書中有詳細的記載。The above pharmaceutical compositions can be prepared according to established pharmaceutical procedures, such as those described in Remington's Pharmaceutical Sciences (17th edition, ed. Alfonoso R. Gennaro, Mack Publishing Company, Easton, Pa (1985).).
在選擇適用於投遞合成胜肽之藥學上可接受賦型劑時，主要需考量此藥學組合物的給藥方式。In selecting a pharmaceutically acceptable excipient suitable for delivery of a synthetic peptide, it is primarily necessary to consider the mode of administration of the pharmaceutical composition.
根據本發明的可任選實施例，可利用關節內注射的方式局部給藥。在此種情形中，可利用無菌水溶液作為藥學上可接受載體，此水溶液較佳為與接受者體液等張的溶液。舉例來說，可將活性成分溶解或懸浮於含有生理可相容物質的水中，所述的生理可相容物質如氯化鈉、甘胺酸及與其相似者，且其pH值經緩衝可與生理條件相容，以得到一水溶液，而後再進行滅菌。可用來製造上述無菌注射溶液或懸浮液的其他稀釋劑或溶劑包括，但不限於，1,3- 丁二醇(1,3-butanediol)、甘露醇(mannitol)、水與林格氏溶液(Ringer’s solution)。也可使用脂肪酸(如，油酸)及其之甘油酯衍生物，或是天然藥學可接受的油(如，橄欖油或菜籽油)來製造可供注射用的溶液或懸浮液。這類油性溶液或懸浮液中也可包含用來稀釋的醇類或羧甲基纖維素(carboxymethyl cellulose)或類似的分散劑。於調製藥學組合物時，亦可使用其他常用的界面活性劑(如，Tweens或Spans系列)、乳化劑，或於製備藥學上可接受劑型時常用的生物可利用性促進劑(bioavailability enhancer)。According to an optional embodiment of the invention, topical administration can be by means of intra-articular injection. In this case, a sterile aqueous solution may be utilized as a pharmaceutically acceptable carrier, and the aqueous solution is preferably a solution which is isotonic with the body fluid of the recipient. For example, the active ingredient may be dissolved or suspended in water containing a physiologically compatible substance such as sodium chloride, glycine, and the like, and the pH thereof may be buffered. The physiological conditions are compatible to obtain an aqueous solution which is then sterilized. Other diluents or solvents which may be employed in the manufacture of such sterile injectable solutions or suspensions include, but are not limited to, 1,3- Butyl diol (1,3-butanediol), mannitol, water and Ringer's solution. Fatty acids (e.g., oleic acid) and their glyceride derivatives, or natural pharmaceutically acceptable oils (e.g., olive oil or rapeseed oil) can also be used to make solutions or suspensions for injectable use. Alcohols or carboxymethyl cellulose or similar dispersing agents for dilution may also be included in such oily solutions or suspensions. Other commonly used surfactants (e.g., Tweens or Spans series), emulsifiers, or bioavailability enhancers commonly used in the preparation of pharmaceutically acceptable dosage forms may also be employed in the preparation of the pharmaceutical compositions.
於可任選的實施例中，所述的藥學組合物可更包含一糖胺聚多醣。糖胺聚多醣是一種線性多醣類，由重複的雙醣(disaccharide)單元所構成，並有一羧基與至少一硫酸。糖胺聚多醣可連接至核心蛋白(core protein)以形成蛋白多醣(proteoglycan)，此乃骨細胞外間質(bone extracellular matrix)的主要成分。在患有骨性關節炎的軟骨處，基質中糖胺聚多醣的含量降低，而使得糖胺聚多醣和第二型膠原蛋白的之間的連結減弱，而使病況加重。因此，提供外來的糖胺聚多醣至病灶部位，有助於治療骨性關節炎。糖胺聚多醣的實施例包括但不限於：玻尿酸與玻尿酸鈉。作為例示而非限制，根據本發明實施例的藥學組合物可包含約1-15%(重量百分比)的玻尿酸。In an optional embodiment, the pharmaceutical composition may further comprise a glycosaminoglycan polysaccharide. The glycosaminoglycan is a linear polysaccharide composed of repeating disaccharide units and having a carboxyl group and at least one sulfuric acid. The glycosaminoglycan polysaccharide can be linked to a core protein to form a proteoglycan, which is a major component of the bone extracellular matrix. In the cartilage with osteoarthritis, the content of glycosaminoglycans in the matrix is lowered, and the link between the glycosaminoglycan polysaccharide and the second type collagen is weakened, and the condition is aggravated. Therefore, providing a foreign glycosaminoglycan to the lesion site helps to treat osteoarthritis. Examples of glycosaminoglycans include, but are not limited to, hyaluronic acid and sodium hyaluronate. By way of illustration and not limitation, a pharmaceutical composition according to embodiments of the invention may comprise from about 1% to about 15% by weight hyaluronic acid.
同樣可任選地，本發明的組合物亦可包含所屬技術領域中具有通常知識者所熟知的藥學上可接受之一或多種添 加劑，以提升合成胜肽的投遞效率、改善使用者的使用經驗。上述可任選的成分包括但不限於一或多種以下成分：乾燥劑(drying Agent)、抗癢劑(anti-itch agents)、抗發泡劑(anti-foaming agents)、緩衝劑(buffers)、中和劑(neutralizing agents)、pH調節劑(pH adjusting agents)、著色劑(coloring agents)、脫色劑(discoloring agents)、軟化劑(emollients)、乳化劑(emulsifying agents)、乳化安定劑(emulsion stabilizers)、增黏劑(viscosity builders)、保濕劑(humectants)、芳香劑(odorants)、防腐劑(preservatives)、抗氧化劑(antioxidants)、化學安定劑(chemical stabilizers)、增稠劑(thickening agents)、硬化劑(stiffening agents)、懸浮劑(suspending agents)等。Also optionally, the compositions of the present invention may also comprise one or more pharmaceutically acceptable ones well known to those of ordinary skill in the art. Adding agents to improve the delivery efficiency of synthetic peptides and improve user experience. The above optional ingredients include, but are not limited to, one or more of the following ingredients: drying agents, anti-itch agents, anti-foaming agents, buffers, Neutralizing agents, pH adjusting agents, coloring agents, discoloring agents, emollients, emulsifying agents, emulsion stabilizers ), viscosity builders, humectants, odorants, preservatives, antioxidants, chemical stabilizers, thickening agents, Stiffening agents, suspending agents, and the like.
在又一態樣中，本發明提出了一種用以治療一個體之骨性關節炎方法。所述個體可以是任何哺乳類動物，包括人類。根據本發明的原理與精神，所述方法能夠促進軟骨再生，進而能夠緩和或治癒該個體的骨性關節炎。In still another aspect, the invention provides a method of treating osteoarthritis in a body. The individual can be any mammal, including a human. In accordance with the principles and spirit of the present invention, the method is capable of promoting cartilage regeneration, which in turn can alleviate or cure osteoarthritis in the individual.
於一實施例中，上述方法包含對該個體投予一有效量的根據本發明上述態樣/實施例之合成胜肽，藉使所述胜肽到達該個體體內鄰近病灶的滑液囊。In one embodiment, the method comprises administering to the individual an effective amount of a synthetic peptide according to the above aspect/embodiment of the invention, such that the peptide reaches a synovial sac adjacent to the lesion in the individual.
於可任選的實施例中，可將所述合成胜肽調製為上述本發明態樣/實施例所述的藥學組合物。In an optional embodiment, the synthetic peptide can be formulated into the pharmaceutical compositions described above in the aspects/embodiments of the invention.
於一實施例中，可將所述合成胜肽或藥學組合物以關節內注射的方式局部遞送至該個體的滑液囊內。In one embodiment, the synthetic peptide or pharmaceutical composition can be delivered topically into the synovial sac of the individual by intra-articular injection.
下文提出多個實驗例來說明本發明的某些態樣，以利本發明所屬技術領域中具有通常知識者實作本發明。不應將這些實施例視為對本發明範圍的限制。據信習知技藝者在閱讀了此處提出的說明後，可在不需過度解讀的情形下，完整利用並實踐本發明。此處所引用的所有公開文獻，其全文皆視為本說明書的一部分。A number of experimental examples are set forth below to illustrate certain aspects of the present invention, and the present invention may be practiced by those of ordinary skill in the art. These examples should not be construed as limiting the scope of the invention. It is believed that the skilled artisan, after reading the description set forth herein, may fully utilize and practice the invention without undue interpretation. All publications cited herein are hereby incorporated by reference in their entirety.
DMEM培養基(Dulbecco’s modified Eagle’s medium)、胎牛血清(fetal bovine serum，簡稱FBS)、0.25%胰蛋白酶(trypsin)、抗生素、TRIzol與Dynabeads皆購自Invitrogen(Carlsbad,CA)。玻尿酸(hyaluronic acid，簡稱HA)、單碘乙酸(mono-iodoacetate，簡稱MIA)、二甲亞碸(dimethyl sulfoxide，簡稱DMSO)、纖維連接蛋白(fibronectin)、Percoll、胰島素(insulin)、氫皮質酮(hydrocortisone)、牛血清白蛋白(bovine serum albumin，簡稱BSA)、5-溴-2'-去氧尿苷(5-bromo-2'-deoxyuridine，簡稱BrdU)、Hoechst 33258染料皆購自Sigma-Aldrich(St.Louis,MO)。抗-BrdU抗體、抗-軟骨蛋白聚醣(anti-aggrecan)抗體與抗-第二型膠原蛋白抗體皆購自GeneTex(台北，台灣)。各種螢光染料-結合二級抗體皆購自BioLegend(San Diego,CA)。鏈蛋白酶(Pronase)與膠 原蛋白酶係購自Roche(Indianapolis,IN)。蘇木色素與伊紅(Hematoxylin and eosin，簡稱H&E)染料購自Merck(Rayway,NJ,USA)。DMEM medium (Dulbecco's modified Eagle's medium), fetal bovine serum (FBS), 0.25% trypsin, antibiotics, TRIzol and Dynabeads were purchased from Invitrogen (Carlsbad, CA). Hyaluronic acid (HA), mono-iodoacetate (MIA), dimethyl sulfoxide (DMSO), fibronectin, Percoll, insulin, hydrocorticosterone (hydrocortisone), bovine serum albumin (BSA), 5-bromo-2'-deoxyuridine (BrdU), Hoechst 33258 dyes were purchased from Sigma- Aldrich (St. Louis, MO). Anti-BrdU antibodies, anti-aggrecan antibodies and anti-type 2 collagen antibodies were purchased from GeneTex (Taipei, Taiwan). Various fluorescent dye-binding secondary antibodies were purchased from BioLegend (San Diego, CA). Protease and glue Proproteinase was purchased from Roche (Indianapolis, IN). Hematoxylin and eosin (H&E) dyes were purchased from Merck (Rayway, NJ, USA).
短合成胜肽(包括29-mer(序列編號：3)、24-mer(序列編號：5)、20-mer(序列編號：6)與18-mer(序列編號：7))係購自GenScript(Piscataway,NJ)，於合成後，將其N端醯化(acetylated)並將C端醯胺化(amidated)以提升安定性，並以質譜儀定性(純度>95%)。以DMSO為溶劑，分別將PEDF衍生合成胜肽(29-mer、24-mer、20-mer或18-mer)製成濃度為5 mM的儲備溶液(stock solution)，並儲存於-20℃中以供後續使用。Short synthetic peptides (including 29-mer (SEQ ID NO: 3), 24-mer (SEQ ID NO: 5), 20-mer (SEQ ID NO: 6) and 18-mer (SEQ ID NO: 7)) were purchased from GenScript. (Piscataway, NJ), after synthesis, its N-terminal is acetylated and the C-terminal is amidated to enhance stability and is characterized by mass spectrometry (purity >95%). The PEDF-derived synthetic peptide (29-mer, 24-mer, 20-mer or 18-mer) was made into a 5 mM stock solution in DMSO as a solvent and stored at -20 °C. For subsequent use.
本說明書所載實施例所用的所有動物皆飼育於有溫度與濕度控制的飼養籠中，飼養溫度約24℃至25℃，光暗循環為12：12小時。試驗過程中提供飲水與標準齧齒類飼料供任食。實驗計畫皆通過馬偕紀念醫院(新北市，台灣)倫理委員會核准，並遵循國家動物保護相關規範。All animals used in the examples contained in this specification were housed in a cage with temperature and humidity control at a temperature of about 24 ° C to 25 ° C and a light dark cycle of 12: 12 hours. Drinking water and standard rodent feed are provided for the meal during the test. The experimental plans were approved by the Ethics Committee of the Ma Rong Memorial Hospital (New Taipei City, Taiwan) and followed the national animal protection regulations.
對大鼠進行MIA關節內注射，以建立骨性關節炎的動物模型。具體來說，10週大的成年雄性Sprague-Dawley大白鼠(平均體重312±11 g)經腹腔內注射若朋(xylazine，用量為每公斤體重10毫克)以進行麻醉；接著在右膝以單一劑關節內注射的方式注入MIA(1毫克MIA溶於25 μl的無菌生理食鹽水中)，注射時，將膝蓋處彎曲成90度， 利用27G的針頭，經由膝韌帶(patellar ligament)注入上述MIA溶液。Rats were intra-articularly injected with MIA to establish an animal model of osteoarthritis. Specifically, a 10-week-old adult male Sprague-Dawley rat (average weight 312 ± 11 g) was anesthetized by intraperitoneal injection of xylazine (10 mg per kilogram body weight); followed by a single knee in the right knee. The intra-articular injection was injected into MIA (1 mg of MIA dissolved in 25 μl of sterile physiological saline), and the knee was bent at 90 degrees when injected. The above MIA solution was injected through a patellar ligament using a 27G needle.
欲偵測活體內細胞擴增時，以DMSO為溶劑，製得含80 mM BrdU的儲備溶液。將150 μl的BrdU儲備溶液和350 μl的磷酸緩衝液(phosphate-buffered saline，簡稱PBS)混合後，經腹膜內注射至大白鼠體內，並於16小時後將大白鼠安樂死。其後根據下文所述方法利用抗-BrdU抗體來標記BrdU，以評估DNA合成之情形。To detect cell expansion in vivo, a stock solution containing 80 mM BrdU was prepared using DMSO as a solvent. 150 μl of BrdU stock solution and 350 μl of phosphate-buffered saline (PBS) were mixed, intraperitoneally injected into rats, and the rats were euthanized 16 hours later. Thereafter, BrdU was labeled with an anti-BrdU antibody according to the method described below to evaluate the situation of DNA synthesis.
自8週大雄性Sprague-Dawley大白鼠的股骨髁(包括前方、後方與側邊等區域)得關節軟骨，取樣時需小心以免滑液膜引起污染並避免破壞骨質結構。取下的樣本切成約0.5 mm3的小片後，依序以鏈蛋白酶(70 U/ml，37℃下1小時)與膠原蛋白酶(300 U/ml，37℃下3小時)處理。之後，以無菌PBS沖洗一次，之後再以添加了2%FBS的DMEM培養基沖洗兩次，以移除膠原蛋白酶。進行初代培養時，在六孔盤上放入經10 μg/ml纖維連接蛋白(fibronectin)塗覆的蓋玻片，並將對錐藍(trypan blue)呈陰性反應的細胞以每孔6000個的密度培養於六孔盤中，所用的培養基為DMEM並添加10%FBS與1%青黴素/鏈黴素抗生素混合物。隔天，將培養基換成標準增殖培養基(standard expansion medium，含有10% FBS-DMEM、0.1 mM維生素C、0.5 mg/ml L-葡萄糖、100 mM HEPES、 1 mM丙酮酸鈉(sodium pyruvate)、2 mM L-麩胺酸與抗生素)，培養基中可添加或不添加50 nM PEDF胜肽。每3天更換新鮮培養基，共培養12天。Articular cartilage was obtained from the femoral condyles (including the anterior, posterior, and lateral regions) of the 8-week male Sprague-Dawley rats. Care should be taken during sampling to avoid contamination of the synovial membrane and to avoid damage to the bone structure. The removed sample was cut into small pieces of about 0.5 mm3 and sequentially treated with chain protease (70 U/ml, 1 hour at 37 ° C) and collagenase (300 U/ml, 3 hours at 37 ° C). Thereafter, it was washed once with sterile PBS, and then washed twice with DMEM medium supplemented with 2% FBS to remove collagenase. For primary culture, a 10 μg/ml fibronectin-coated coverslip was placed on a six-well plate, and cells with a negative reaction to trypan blue were 6000 per well. Density was cultured in a six-well plate using DMEM in a mixture of 10% FBS and 1% penicillin/streptomycin antibiotic. The next day, the medium was changed to a standard expansion medium containing 10% FBS-DMEM, 0.1 mM vitamin C, 0.5 mg/ml L-glucose, 100 mM HEPES, 1 mM sodium pyruvate, 2 mM L-glutamic acid and antibiotics, 50 nM PEDF peptide may or may not be added to the medium. Fresh medium was changed every 3 days for a total of 12 days.
8週大雄性Sprague-Dawley大鼠經腹腔內注射若朋(xylazine，用量為每公斤體重10毫克)以進行麻醉。在無菌環境下移出股骨，以PBS和抗生素的混合物沖洗約5分鐘後，切除所有軟組織、將其股端(epiphysis)截斷，之後以肝素和DMEM混合物反覆沖洗骨髓腔。將收集到的骨髓細胞以1000×g離心10分鐘，而後將細胞沈澱物重新溶於DMEM培養基中，並將此細胞懸浮液轉移到含有5毫升Percoll(1.073 g/ml)的15-ml離心管中，以1500×g離心30分鐘。之後取出位於中間層的單核細胞(mononuclear cells)，以PBS沖洗三次後，重新懸浮於低葡萄糖含量的DMEM，(添加10%經熱不活化的FBS與1%青黴素/鏈黴素混合物)。在95%空氣和5%二氧化碳以及37℃的環境下培養2週，並每4天更換新鮮培養基。棄置未貼附的細胞，並保存貼附細胞。在培養約一週後，初代間葉幹細胞(mesenchymal stem cells，簡稱MSCs)會長至約80-90%滿的狀態。Eight-week-old male Sprague-Dawley rats were anesthetized by intraperitoneal injection of xylazine (10 mg/kg body weight). The femur was removed in a sterile environment, rinsed with a mixture of PBS and antibiotics for about 5 minutes, all soft tissue was excised, its epiphysis was truncated, and then the medullary cavity was repeatedly rinsed with a mixture of heparin and DMEM. The collected bone marrow cells were centrifuged at 1000 x g for 10 minutes, and then the cell pellet was redissolved in DMEM medium, and the cell suspension was transferred to a 15-ml centrifuge tube containing 5 ml of Percoll (1.073 g/ml). Centrifuge at 1500 x g for 30 minutes. The mononuclear cells in the middle layer were then removed, washed three times with PBS, and resuspended in low glucose DMEM (addition of 10% heat inactivated FBS to 1% penicillin/streptomycin mixture). Incubate for 2 weeks in an environment of 95% air and 5% carbon dioxide and 37 ° C, and replace the fresh medium every 4 days. Discard the unattached cells and save the attached cells. After about one week of culture, the primary mesenchymal stem cells (MSCs) will grow to a state of about 80-90% full.
欲引發間葉幹細胞分化為軟骨細胞時，將5 X 105 的細胞培養於促進軟骨細胞生成的培養基(chondrogenic medium)中，此培養基為高葡萄糖含量的DMEM培養基， 含有100 nM甲基脫氫皮質固醇(dexamethasone)、0.17 mM維生素C-2磷酸鹽(ascorbic acid-2 phosphate)、10 μg/ml胰島素、5 μg/ml運鐵蛋白(transferrin)、5 ng/ml硒、1 mM丙酮酸鈉、2 mM L-麩胺酸與2% FBS)，並添加10 ng/mlTGF-β2(R&D Systems；Minneapolis,MN)。在接受PEDF處理的組別中，將細胞培養於上述促進軟骨細胞生成的培養基中，並添加50 nM的PEDF胜肽。每兩天更換一次培養基，培養一週。To induce mesenchymal stem cells to differentiate into chondrocytes, 5×10 5 cells were cultured in a chondrogenic medium that promotes chondrocyte production in a high glucose content DMEM medium containing 100 nM methyl dehydrogenated cortex. Dexamethasone, 0.17 mM ascorbic acid-2 phosphate, 10 μg/ml insulin, 5 μg/ml transferrin, 5 ng/ml selenium, 1 mM sodium pyruvate 2 mM L-glutamic acid with 2% FBS) and 10 ng/ml TGF-β2 (R&D Systems; Minneapolis, MN). In the group subjected to PEDF treatment, the cells were cultured in the above-mentioned medium for promoting chondrocyte production, and 50 nM of PEDF peptide was added. The medium was changed every two days and cultured for one week.
切下膝關節後，移除周圍軟組織，將樣本以4%甲醛固定後，利用Shandon TBD-2脫鈣劑(decalcifier，購自Thermo Scientific；Logan,UT)處理。之後沿著關節中央徑向(mid-sagittally)切開，再以固態石蠟(paraffin blocks)包埋。沿縱向將組織切成厚度約5-μm的薄片。After the knee joint was cut, the surrounding soft tissue was removed, and the sample was fixed with 4% formaldehyde and treated with a Shandon TBD-2 decalcifier (available from Thermo Scientific; Logan, UT). It is then cut mid-sagittally along the center of the joint and then embedded in solid paraffin blocks. The tissue was cut into a sheet having a thickness of about 5-μm in the longitudinal direction.
於使用前，於二甲苯(xylene)中移除石蠟，並以一系列濃度梯度的乙醇回復樣本。之後，可利用H&E染色進行一般組織學觀察，或進行螢光染色。每個膝蓋樣本中取出20個切片，以盡可能涵蓋退化最嚴重的部分。The paraffin was removed in xylene before use and the sample was returned to the series with a concentration gradient of ethanol. Thereafter, general histological observations or fluorescent staining can be performed using H&E staining. Take 20 slices from each knee sample to cover the most degraded parts as much as possible.
經石蠟包埋的關節樣本於二甲苯(xylene)中移除石蠟，並以一系列濃度梯度的乙醇回復樣本。脫蠟的樣本在室溫下以1 N HCl處理1小時，而後再進行免疫螢光染色。Paraffin-embedded joint samples were removed from paraffin in xylene and samples were returned to a series of concentrations of ethanol. The dewaxed sample was treated with 1 N HCl for 1 hour at room temperature and then subjected to immunofluorescence staining.
進行免疫染色分析前，將脫臘的組織切片以10%山羊血清和5%的BSA處理約1小時以進行阻斷。之後以抗-軟骨蛋白聚醣(稀釋比1：100)、抗-第二型膠原蛋白(稀釋比1：100)與抗-BrdU(稀釋比1：100)的一級抗體在37℃下培育2小時，再和與適當螢光染劑結合之驢IgG抗體於室溫下培育1小時。之後將樣本和Hoechst 33258染劑接觸約7分鐘，以進行細胞核的對比染色(counter staining)，經染色的細胞核呈現藍色。Prior to immunostaining analysis, de-waxed tissue sections were treated with 10% goat serum and 5% BSA for about 1 hour to block. Then, the primary antibody with anti- cartilage proteoglycan (dilution ratio 1:100), anti-type 2 collagen (dilution ratio 1:100) and anti-BrdU (dilution ratio 1:100) was incubated at 37 ° C. The IgG antibody was incubated with the appropriate fluorescent dye for 1 hour at room temperature. The sample was then contacted with Hoechst 33258 dye for about 7 minutes for counter staining of the nuclei, and the stained nuclei appeared blue.
使用Zeiss螢光顯微鏡(Zeiss epifluorescence microscope)來擷取樣本影像。計算細胞數目時，從每一個樣本中隨機選取20個視野，由不知道實驗分組的人員人力計算，並重複三次。The image was sampled using a Zeiss epifluorescence microscope. When calculating the number of cells, 20 fields of view were randomly selected from each sample, calculated by the person who did not know the experimental group, and repeated three times.
反轉錄聚合酶連鎖反應(reverse-transcription polymerase chain reaction，簡稱RT-PCR)分析方法如下。利用TRIzol自細胞內萃取出全RNA，並以不含核糖核酸水解酶(RNase-free)的第一型去氧核糖核酸水解酶(DNase I)(Qiagen,Santa Clarita,CA)處理以移除基因組DNA，之後以RNA純化套組Dynabeads進行純化。在20 μl的反應緩衝液(含0.25 μg的隨機引子與0.8 mM去氧核糖核苷三磷酸鹽(dNTPs))中加入1 μg取自BM-MSC的全RNA與200單位的反轉錄酶(Roche,Mannheim,Germany)，於42℃下反應1小時，以將RNA反轉錄為cDNA。The reverse-transcription polymerase chain reaction (RT-PCR) analysis method is as follows. Total RNA was extracted from cells by TRIzol and treated with RNase-free type 1 deoxyribonuclease (DNase I) (Qiagen, Santa Clarita, CA) to remove the genome DNA was then purified using RNA purification kit Dynabeads. Add 1 μg of total RNA from BM-MSC to 200 units of reverse transcriptase in 20 μl of reaction buffer (containing 0.25 μg of random primer and 0.8 mM deoxyribonucleoside triphosphate (dNTPs)) (Roche , Mannheim, Germany), reacted at 42 ° C for 1 hour to reverse transcribe RNA into cDNA.
在後續的PCR反應中，取2 μl的cDNA作為反應模板。PCR反應的反應體積為30 μl，其中含有15 μl的EconoTaq® PLUS GREEN 2× Master Mix(Lucigen® Corp.)、1 μM的各種引子以及2 μl的模板DNA。合成cDNA的增殖反應約18-22個循環(變性：20秒、94℃；黏合：30秒、57℃；以及聚合：40秒、72℃)。每種引子組所用的循環數落在擴充的線性範圍內。用以增殖大白鼠軟骨蛋白聚醣(aggregan)基因(註冊號：J03485)的引子組包含正向引子(TTGGAAATCCAGAACCTTCG；序列編號：12)與反向引子(GTCCAGTGTGTAGCGTGTGG；序列編號：13)，其PCR產物大小約149 bp。另外以大白鼠甘油醛3-磷酸去氫酶(glyceraldehyde 3-phosphate dehydrogenase，簡稱GAPDH)基因(註冊號：X02231.1)作為管家基因(housekeeping gene)，以將基因表現量標準化。用以增殖GAPDH基因的引子組包括正向引子(AGACAGCCGCATCTTCTTGT；序列編號：14)與反向引子(CTTGCCGTGGGTAGAGTCAT；序列編號：15)，而PCR產物的大小約207 bp。利用含有溴化乙錠(ethidium bromide)的2%洋菜膠對PCR產物進行電泳分析，並以UV光照使其顯影。利用FUJI LAS-3000系統與Multi Gauge Ver.1.01軟體(Fujifilm,Tokyo,Japan)對PCR產物進行光密度法定量分析。In the subsequent PCR reaction, 2 μl of cDNA was taken as a reaction template. The PCR reaction has a reaction volume of 30 μl containing 15 μl of EconoTaq® PLUS GREEN 2× Master Mix (Lucigen® Corp.), 1 μM of various primers, and 2 μl of template DNA. The proliferative reaction of the synthetic cDNA was about 18-22 cycles (denaturation: 20 seconds, 94 ° C; adhesion: 30 seconds, 57 ° C; and polymerization: 40 seconds, 72 ° C). The number of cycles used for each primer set falls within the linear range of the extension. The primer set used to proliferate the rat aggregan gene (registration number: J03485) contains a forward primer (TTGGAAATCCAGAACCTTCG; SEQ ID NO: 12) and a reverse primer (GTCCAGTGTGTAGCGTGTGG; SEQ ID NO: 13), and the PCR product thereof The size is about 149 bp. In addition, the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene (registration number: X02231.1) was used as a housekeeping gene to standardize gene expression. The primer set used to propagate the GAPDH gene includes a forward primer (AGACAGCCGCATCTTCTTGT; SEQ ID NO: 14) and a reverse primer (CTTGCCGTGGGTAGAGTCAT; SEQ ID NO: 15), and the PCR product has a size of about 207 bp. The PCR product was electrophoresed using 2% acacia gum containing ethidium bromide and developed by UV light. The PCR products were quantitatively analyzed by optical density method using a FUJI LAS-3000 system and Multi Gauge Ver. 1.01 software (Fujifilm, Tokyo, Japan).
將結果表示為平均值±平均值的標準差(standard error of the mean，SEM)。利用單向(one-way)ANOVA分析來進行統計比較。除非另有說明，P <0.05時具有統計上的顯著差異。The results are expressed as mean ± standard error of the mean (SEM). Statistical comparisons were made using one-way ANOVA analysis. Statistically significant differences were obtained at P < 0.05 unless otherwise stated.
膝蓋的骨性關節炎是一種常見的慢性退化性疾病，其特徵在於關節軟骨的減損。單碘乙酸是一種糖解(glycolysis)抑制劑，已知將MIA注射至鼠類的股脛關節空間(femorotibial joint space)會引發關節軟骨的流失，此過程與人類骨性關節炎的症狀相近。研究顯示，在注射MIA後7天，通常會出現嚴重的軟骨細胞退化與壞死等情形。因而，本實驗例中，在MIA注射後第8天開始進行PEDF治療，以探究此處提出的PEDF胜肽是否可促進軟骨再生。Osteoarthritis of the knee is a common chronic degenerative disease characterized by impairment of articular cartilage. Monoiodoacetic acid is a glycolysis inhibitor. It is known that injection of MIA into the femorotibial joint space of rats causes the loss of articular cartilage, which is similar to the symptoms of human osteoarthritis. Studies have shown that 7 days after the injection of MIA, severe chondrocyte degeneration and necrosis usually occur. Thus, in this experimental example, PEDF treatment was started on the 8th day after MIA injection to investigate whether the PEDF peptide proposed herein promotes cartilage regeneration.
將MIA注射後的大白鼠隨機分派至六個實驗群組，每組6隻，並分別接受以下處理。HA組的大白鼠接受25 μl濃度為5%的玻尿酸注射。在載體/HA組中，將DMSO載體溶於25 μl的5%玻尿酸中，而後注射至大白鼠體內。在29-mer/HA、24-mer/HA、20-mer/HA與18-mer/HA等組別中，將0.2 mM PEDF胜肽(29-mer、24-mer、20-mer或18-mer)溶於25 μl的5%玻尿酸中，而後注射至大白鼠體內。以上處理皆透過單一劑關節內注射，分別在MIA注射後第8、12、16與20天注射一次。MIA-injected rats were randomly assigned to six experimental groups of 6 animals each and received the following treatments. Rats in the HA group received 25 μl of a 5% hyaluronic acid injection. In the vehicle/HA group, the DMSO carrier was dissolved in 25 μl of 5% hyaluronic acid and then injected into the rats. In the 29-mer/HA, 24-mer/HA, 20-mer/HA and 18-mer/HA groups, 0.2 mM PEDF peptide (29-mer, 24-mer, 20-mer or 18-) Mer) is dissolved in 25 μl of 5% hyaluronic acid and then injected into rats. All of the above treatments were injected intra-articularly through a single dose, and were injected once on the 8th, 12th, 16th and 20th day after MIA injection.
在MIA注射後第18天與第25天取得關節樣本，經H&E染色後的代表性照片如第1圖所示。將載體/HA組和18-mer/HA組和未接受MIA注射的正常關節(正常組)相比較，可以發現到MIA注射會導致和骨性關節炎類似的關節形態改變，包括軟骨組織變小、軟骨表面纖維化以及軟骨下骨(subchondral bone)崩壞等，這些變化在承受體重的部位尤其明顯。相較於載體/HA組和18-mer/HA組，在接受20-mer/HA處理的組別中，軟骨表面較為平滑，且軟骨和軟骨下骨相對較為完整。其他PEDF胜肽諸如29-mer與24-mer處理(資料未示出)亦可產生和20-mer類似的效果。這些結果顯示此處提出的PEDF胜肽可以改善與骨性關節炎相關的病理學特徵。A joint sample was obtained on the 18th day and the 25th day after the MIA injection, and a representative photograph after H&E staining is shown in Fig. 1. Comparing the vehicle/HA group with the 18-mer/HA group and the normal joint (normal group) not receiving MIA injection, it was found that MIA injection resulted in joint morphology changes similar to osteoarthritis, including cartilage tissue becoming smaller. , cartilage surface fibrosis and subchondral bone collapse, etc., these changes are particularly evident in the site of weight bearing. Compared to the vehicle/HA group and the 18-mer/HA group, the cartilage surface was smoother and the cartilage and subchondral bone were relatively intact in the 20-mer/HA treated group. Other PEDF peptides such as 29-mer and 24-mer treatment (data not shown) can also produce similar effects as 20-mer. These results show that the PEDF peptide proposed herein can improve the pathological features associated with osteoarthritis.
正常軟骨是層狀結構的組織，根據功能和結構可將其分成表層區(superficial zone)、中層區(middle zone；又稱過渡區(transitional zone))以及深層區(deep zone；又稱徑向區(radial))等三層。當膝關節活動時表層區的平滑表層提供足夠的潤滑度用於緩衝應力；本區約佔整個關節軟骨厚度的10%至20%。中層區約佔關節軟骨的40%至60%，其細胞分泌蛋白多醣和膠原纖維，以提供足夠的支撐力。深層區約佔軟骨的30%，主要是由直徑較大的膠原蛋白纖維所組成，其排列方向與關節表面垂直對抗剪應力。表層區中的軟骨細胞多成狹長紡錘狀，膠原蛋白纖維的排列非常規則，且與關節面平行。中層區中膠原蛋白的白列較不規則，且軟骨細胞外型較圓(相較於表層中的軟 骨細胞)。深層區中的軟骨細胞通常成條狀(columnar)，且和膠原蛋白纖維的方向平行並垂直於關節線。在第2圖的代表性照片中，可以觀察到此種層狀構造，所示照片係取自MIA注射後25天的膝關節樣本。Normal cartilage is a layered structure that can be divided into a superficial zone, a middle zone (also called a transitional zone), and a deep zone (also called a radial zone) according to its function and structure. The area (radial) and other three layers. The smooth surface layer of the superficial region provides sufficient lubrication for cushioning stress when the knee is active; this region accounts for approximately 10% to 20% of the thickness of the entire articular cartilage. The middle zone accounts for about 40% to 60% of articular cartilage, and its cells secrete proteoglycans and collagen fibers to provide sufficient support. The deep zone accounts for about 30% of the cartilage, and is mainly composed of larger diameter collagen fibers, which are arranged in a direction perpendicular to the joint surface against shear stress. The chondrocytes in the superficial region are mostly elongated and spindle-shaped, and the arrangement of collagen fibers is very regular and parallel to the articular surface. The white column of collagen in the middle layer is more irregular, and the cartilage cell appearance is rounder (compared to the softness in the surface layer) Bone cells). The chondrocytes in the deep zone are usually in the form of a columnar and parallel to the direction of the collagen fibers and perpendicular to the joint line. In the representative photograph of Fig. 2, such a layered structure was observed, and the photographs shown were taken from a knee joint sample 25 days after the MIA injection.
由第2圖的照片可以看出，正常軟骨中的細胞數目較低(hypoccllular)；而經過載體/HA處理的膝蓋中，可以觀察到表層區中的軟骨細胞大量流失，且在中層區與深層區中，可以看到有退化細胞聚集成群(如箭頭所指處)。相較之下，在29-mer/HA與20-mer/HA組中，可以看到整個軟骨內皆有大量新生成的軟骨細胞。此外，在經過此處提出之PEDF胜肽處理的膝蓋中，每一層中的軟骨細胞呈現較規則的排列。一般來說，軟骨再生的過程包含軟骨細胞增殖以及結構重組兩個部分；由此可知，此處提出的PEDF胜肽可促進軟骨再生過程。It can be seen from the photograph in Fig. 2 that the number of cells in normal cartilage is low (hypoccllular); while in the carrier/HA treated knee, a large amount of chondrocytes in the superficial region can be observed, and in the middle and deep layers. In the district, you can see that there are degenerating cells clustered together (as indicated by the arrow). In contrast, in the 29-mer/HA and 20-mer/HA groups, a large number of newly formed chondrocytes were observed throughout the cartilage. In addition, in the knee treated with the PEDF peptide treatment proposed herein, the chondrocytes in each layer exhibited a relatively regular arrangement. In general, the process of cartilage regeneration involves both chondrocyte proliferation and structural recombination; thus, it is known that the PEDF peptide proposed herein can promote the cartilage regeneration process.
骨性關節炎會導致軟骨退化引起軟骨內細胞間質(如軟骨蛋白聚醣與第二型膠原蛋白)的流失。如第3圖左方照片所示，在經過20-mer處理的軟骨細胞中，其表層區、中層區與深層區內皆可看到大量對軟骨蛋白聚醣呈陽性反應的信號(綠色)；相較之下，在經過載體處理的軟骨中，僅可觀察到微弱的螢光信號。利用抗-第二型膠原蛋白抗體來染色膝關節切片，亦可觀察到相似的結果(資料未示出)。即此處提出的PEDF胜肽有助於保持細胞外間質的含量，此一結果與此處提出之PEDF胜肽可促進軟骨修復的論點相呼應。此外，利用軟骨蛋白聚醣染色亦可觀察軟骨 細胞的族群大小，結果顯示經20-mer處理之軟骨內，軟骨細胞的數目高於經載體處理的軟骨。Osteoarthritis can lead to the loss of cartilage and the loss of intracellular chondrocytes (such as cartilage proteoglycans and type 2 collagen). As shown in the photo on the left of Figure 3, in the 20-mer-treated chondrocytes, a large number of signals positive for cartilage proteoglycans (green) were observed in the surface layer, the middle layer and the deep layer; In contrast, in the carrier-treated cartilage, only weak fluorescent signals were observed. Similar results were observed by staining the knee joint with an anti-type 2 collagen antibody (data not shown). That is, the PEDF peptide proposed here helps to maintain the extracellular matrix content, and this result echoes the argument that the PEDF peptide proposed herein can promote cartilage repair. In addition, cartilage proteoglycan staining can also observe cartilage The population size of the cells showed that the number of chondrocytes in the cartilage treated with 20-mer was higher than that of the vehicle-treated cartilage.
對軟骨蛋白聚醣與細胞核(以Hoechst 33258染料染成藍色)進行雙重免疫螢光染色，代表性照片如第3圖中間所示；結果顯示，在20-mer/HA組中，有超過95%的細胞對軟骨蛋白聚醣呈陽性反應；意味著此處提出的PEDF胜肽能夠藉由引發軟骨細胞擴增而促進軟骨癒合。Double immunofluorescence staining of cartilage proteoglycans and nuclei (blue stained with Hoechst 33258 dye), representative photographs are shown in the middle of Figure 3; results show that in the 20-mer/HA group, there are more than 95 % of cells are positive for cartilage proteoglycans; meaning that the PEDF peptide proposed here can promote cartilage healing by triggering chondrocyte expansion.
此外，利用抗-BrdU抗體進行免疫組織分析，結果顯示相較於載體/HA組，在20-mer/HA組中，BrdU-陽性細胞的數目較多(第3圖右方照片)。根據上文所述的方法，於每一個膝關節樣本中取三個切片(每組6隻大白鼠)，以進行定量分析。以BrdU/軟骨蛋白聚醣標記指數(%)來表示對BrdU和軟骨蛋白聚醣皆呈陽性之細胞，其計算方式係將BrdU-與軟骨蛋白聚醣-雙陽性細胞的數目除以軟骨蛋白聚醣-陽性細胞數，並以百分比表示之。定量分析結果摘要整理於表1；*P <0.0001相較於載體/HA組。Further, immunohistochemical analysis using an anti-BrdU antibody revealed that the number of BrdU-positive cells was larger in the 20-mer/HA group than in the vehicle/HA group (photograph of the right in Fig. 3). Three sections (6 rats per group) were taken from each knee sample for quantitative analysis according to the method described above. Cells expressing both BrdU and cartilage proteoglycans were expressed by BrdU/catenin marker index (%), calculated by dividing the number of BrdU- and cartilage proteoglycan-double positive cells by cartilage protein aggregation The number of sugar-positive cells, expressed as a percentage. A summary of the quantitative analysis results is summarized in Table 1; * P < 0.0001 compared to the vehicle/HA group.
表1的數據指出以此處提出的PEDF胜肽(如，29-mer、24-mer與20-mer)進行處理，可促進軟骨細胞擴增(相較於HA組或載體/HA組)。亦可注意到，不含「SLGA殘基」的18-mer則不具有所述的促進效果。The data in Table 1 indicates that treatment with the PEDF peptides presented herein (eg, 29-mer, 24-mer, and 20-mer) promotes chondrocyte expansion (as compared to the HA group or the vehicle/HA group). It can also be noted that the 18-mer which does not contain the "SLGA residue" does not have the promoting effect described above.
軟骨細胞擴增為軟骨再生的重要機制。總結來說，實驗例1的結果顯示此處提出的PEDF胜肽能夠有效促進受損軟骨中的軟骨細胞擴增。因而，此處提出的PEDF胜肽可促進軟骨再生。Chondrocyte expansion is an important mechanism for cartilage regeneration. In summary, the results of Experimental Example 1 show that the PEDF peptide proposed herein is effective in promoting chondrocyte expansion in damaged cartilage. Thus, the PEDF peptide proposed herein promotes cartilage regeneration.
由大白鼠關節軟骨分離出細胞，並培養於添加或不添加PEDF胜肽的培養基中，以探究此處提出的PEDF胜肽是否可刺激軟骨細胞擴增。Cells were isolated from rat articular cartilage and cultured in medium with or without PEDF peptide to explore whether the PEDF peptide proposed herein stimulates chondrocyte expansion.
第4A圖的照片顯示出當培養基中含有此處提出的PEDF胜肽時，軟骨細胞的生長速度較快，且會聚集成較大的聚落。相較之下，在不含PEDF胜肽的載體組中，軟骨細胞聚落較小。另外，免疫染色分析顯示聚落為軟骨蛋白聚醣陽性，確認聚落中細胞主要是軟骨細胞，結果如第4A圖***的照片所示。關節軟骨分離出的細胞在單層培養系統中隨機選取20個視野來定量對軟骨蛋白聚醣呈陽性的細胞數目(第4B圖)，並以軟骨蛋白聚醣標記指數(%)來表示，此數值是將經軟骨蛋白聚醣標記的細胞數目除以細胞總數(以Hoechst 33258染料標記細胞核)並以百分比 表示。定量分析結果摘要整理於表2；*P <0.01相較於未處理組。The photograph of Fig. 4A shows that when the culture medium contains the PEDF peptide as proposed herein, the growth rate of chondrocytes is faster and the aggregation is integrated into larger colonies. In contrast, chondrocyte colonies were smaller in the vector group containing no PEDF peptide. In addition, immunostaining analysis showed that the colonies were positive for cartilage proteoglycans, and it was confirmed that the cells in the colonies were mainly chondrocytes, and the results were as shown in the photograph inserted in Fig. 4A. The cells isolated from articular cartilage were randomly selected from the monolayer culture system to quantify the number of cells positive for cartilage proteoglycan (Fig. 4B) and expressed by the cartilage proteoglycan index (%). Values are the number of cells labeled with cartilage proteoglycan divided by the total number of cells (labeled nuclei with Hoechst 33258 dye) and expressed as a percentage. A summary of the quantitative analysis results is summarized in Table 2; * P < 0.01 compared to the untreated group.
總結來看，此處提出之PEDF胜肽(如29-mer、24-mer與20-mer)可促進培養系統內的軟骨細胞擴增；此一結果可支持上文實驗例1的結論，即，此處提出的PEDF胜肽可於活體內刺激軟骨細胞擴增。此外，經培養之軟骨細胞中軟骨蛋白聚醣含量較高，亦可與上文所述的活體內軟骨基質再生之結果相呼應。In conclusion, the PEDF peptides proposed here (such as 29-mer, 24-mer and 20-mer) can promote the expansion of chondrocytes in the culture system; this result can support the conclusion of Experimental Example 1 above, namely The PEDF peptide proposed here can stimulate chondrocyte expansion in vivo. In addition, the high content of cartilage proteoglycan in the cultured chondrocytes can also correspond to the results of the regeneration of the cartilage matrix in vivo described above.
在軟骨修復過程中，間葉幹細胞被認為是產生軟骨細胞的來源。本實驗例的目的再探究此處提出的PEDF胜肽是否可促進培養中的間葉幹細胞分化為軟骨細胞。Mesenchymal stem cells are thought to be the source of chondrocytes during cartilage repair. The purpose of this experimental example is to explore whether the PEDF peptide proposed here can promote the differentiation of mesenchymal stem cells into chondrocytes in culture.
根據上文「材料與方法」一節所述的步驟來分離並培養間葉幹細胞，並利用RT-PCR分析來定量軟骨蛋白聚醣mRNA的表現量，所得結果以GAPDH基因的表現量進行標 準化。將載體組的表現率設定為1，以計算其他組別中mRNA表現量相對於載體組的誘導率(fold of induction)，結果摘要整理於表3；*P <0.0002相較於載體組。Mesenchymal stem cells were isolated and cultured according to the procedure described in the section "Materials and Methods" above, and the expression amount of cartilage proteoglycan mRNA was quantified by RT-PCR analysis, and the results were normalized by the amount of GAPDH gene expression. The expression rate of the carrier group was set to 1 to calculate the fold of the mRNA expression in the other groups relative to the carrier group, and the results are summarized in Table 3; * P < 0.0002 compared to the carrier group.
由表3可以看出，培養於標準增殖培養基中的間葉幹細胞內幾乎無法偵測到軟骨蛋白聚醣mRNA的表現。相反地，當培養於促進軟骨細胞生成之培養基內時，有大量的間葉幹細胞分化為軟骨細胞，而能夠觀察到大量的軟骨蛋白聚醣mRNA表現(載體組)。若將細胞培養於含有此處所述之PEDF胜肽(如29-mer、24-mer與20-mer)的培養基中，則會使得軟骨蛋白聚醣mRNA的表現量至少提高為載體組的3.5倍。此外，以18-mer(即，不含所述SLGA殘基的PEDF胜肽)處理時，則無法促進間葉幹細胞朝向軟骨細胞分化。As can be seen from Table 3, the expression of cartilage proteoglycan mRNA was hardly detected in mesenchymal stem cells cultured in standard proliferation medium. Conversely, when cultured in a medium that promotes chondrocyte production, a large number of mesenchymal stem cells differentiate into chondrocytes, and a large amount of cartilage proteoglycan mRNA expression (carrier group) can be observed. If cells are cultured in a medium containing the PEDF peptides described herein (eg, 29-mer, 24-mer, and 20-mer), the amount of expression of the cartilage proteoglycan mRNA is increased to at least 3.5 of the vector group. Times. Furthermore, when treated with 18-mer (i.e., PEDF peptide without the SLGA residue), it was not possible to promote mesenchymal stem cells to differentiate into chondrocytes.
亦對細胞進行軟骨蛋白聚醣與第二型膠原蛋白的染色，以探討此處提出之PEDF胜肽於促進分化的活性。隨機選取20個視野後，定量軟骨蛋白聚醣-陽性細胞，並以軟骨蛋白聚醣標記指數(%)來表示，其計算方式是將經 軟骨蛋白聚醣標記的細胞除以細胞總數(以Hoechst 33258染料標記細胞核)，再以百分比表示。結果摘要整理於表4；*P <0.0001相較於載體組。The cells were also stained with cartilage proteoglycan and type 2 collagen to investigate the activity of the PEDF peptide proposed herein to promote differentiation. After 20 fields of view were randomly selected, the cartilage proteoglycan-positive cells were quantified and expressed by the cartilage proteoglycan index (%), which was calculated by dividing the cartilage proteoglycan-labeled cells by the total number of cells (in Hoechst). 33258 dye labeled nuclei), expressed as a percentage. The results are summarized in Table 4; * P < 0.0001 compared to the vehicle group.
表4的結果顯示，當培養基中加入此處提出的PEDF胜肽(如，29-mer、24-mer與20-mer)時，會有更多細胞分化為軟骨細胞(相較於載體組)。相較之下，18-mer則無法提供這種促進間葉幹細胞分化為軟骨細胞的效果。總結來看，這些結果顯示此處提出的PEDF胜肽可促進間葉幹細胞朝向軟骨細胞分化。The results in Table 4 show that when the PEDF peptide (eg, 29-mer, 24-mer, and 20-mer) proposed herein was added to the medium, more cells differentiated into chondrocytes (compared to the vehicle group). . In contrast, 18-mer is unable to provide this effect of promoting mesenchymal stem cells to differentiate into chondrocytes. In summary, these results show that the PEDF peptide proposed here promotes the differentiation of mesenchymal stem cells toward chondrocytes.
本揭示內容首度證實短的PEDF合成胜肽對於骨性關節炎所導致的軟骨損傷有保護效果。相較於先前技術通常對個體投予可表現全長PEDF胜肽的載體，此處提出的短、合成PEDF胜肽在使用上不但較為安全，且成本也相對低廉。The present disclosure demonstrates for the first time that a short PEDF synthetic peptide has a protective effect on cartilage damage caused by osteoarthritis. The short, synthetic PEDF peptides presented herein are not only safer but also relatively inexpensive to use, as compared to prior art techniques for the administration of a full length PEDF peptide.
雖然上文實施方式中揭露了本發明的具體實施例，然其並非用以限定本發明，本發明所屬技術領域中具有通常知識者，在不悖離本發明之原理與精神的情形下，當可對其進行各種更動與修飾，因此本發明之保護範圍 當以附隨申請專利範圍所界定者為準。Although the embodiments of the present invention are disclosed in the above embodiments, the present invention is not intended to limit the invention, and the present invention may be practiced without departing from the spirit and scope of the invention. Various modifications and modifications can be made thereto, and thus the scope of protection of the present invention This is subject to the definition of the scope of the accompanying application.
為讓本發明的上述與其他目的、特徵、優點與實施例能更明顯易懂，所附圖式之說明如下：第1圖包含於本發明一實驗例中，經H&E染色之膝關節樣本的代表性照片(F：股骨髁(femoral condyle)；T脛骨髁：(tibial condyle)；M：半月軟骨(meniscus))；第2圖為第1圖所示之膝關節組織切片的代表性顯微照片(原始倍率：400倍)；第3圖包含於本發明另一實驗例中，經免疫染色之膝關節樣本的代表性照片(原始倍率：1000倍)；以及第4圖包含於本發明又一實驗例中，經免疫染色之大鼠關節軟骨細胞的代表性照片(右圖原始倍率：1000倍)。The above and other objects, features, advantages and embodiments of the present invention will become more apparent and understood. The description of the drawing is as follows: Figure 1 is included in an experimental example of the present invention, the H&E stained knee joint sample Representative photographs (F: femoral condyle; T-bone condyle; M: meniscus); Figure 2 is a representative micrograph of the knee joint tissue section shown in Figure 1. Photograph (original magnification: 400 times); Fig. 3 is a representative photograph of an immunostained knee joint sample (original magnification: 1000 times) in another experimental example of the present invention; and Fig. 4 is included in the present invention A representative photograph of immunostained rat articular chondrocytes in an experimental example (original magnification on the right: 1000 times).
<110> 財團法人台灣基督長老教會馬偕紀念社會事業基金會馬偕紀念醫院<110> Taiwan's Presbyterian Church, Ma Rong Memorial Social Enterprise Foundation, Ma Rong Memorial Hospital
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<213> 人工序列<213> Artificial sequence
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<223> 引子<223> Introduction
<400> 13 <400> 13
<210> 14<210> 14
<211> 20<211> 20
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 引子<223> Introduction
<400> 14 <400> 14
<210> 15<210> 15
<211> 20<211> 20
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 引子<223> Introduction
<400> 15 <400> 15
Claims (7)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| TW101134579A TWI491407B (en) | 2012-09-20 | 2012-09-20 | Use of pedf-derived polypeptides for treating osteoarthritis |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| TW101134579A TWI491407B (en) | 2012-09-20 | 2012-09-20 | Use of pedf-derived polypeptides for treating osteoarthritis |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| TW201412327A TW201412327A (en) | 2014-04-01 |
| TWI491407B true TWI491407B (en) | 2015-07-11 |
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| Application Number | Title | Priority Date | Filing Date |
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| TW101134579A TWI491407B (en) | 2012-09-20 | 2012-09-20 | Use of pedf-derived polypeptides for treating osteoarthritis |
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| TW (1) | TWI491407B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2021521115A (en) * | 2018-04-08 | 2021-08-26 | ブリム バイオテクノロジー インクＢｒｉｍ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ， Ｉｎｃ． | Use of PEDF-derived short-chain peptides in the treatment of osteoarthritis |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005041887A2 (en) * | 2003-10-29 | 2005-05-12 | The Johns Hopkins University | Pigment epithelium-derived factor, novel biological activity and methods of use |
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2012
- 2012-09-20 TW TW101134579A patent/TWI491407B/en active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005041887A2 (en) * | 2003-10-29 | 2005-05-12 | The Johns Hopkins University | Pigment epithelium-derived factor, novel biological activity and methods of use |
Non-Patent Citations (2)
| Title |
|---|
| D Pfander et al., "Pigment epithelium derived factor—the product of the EPC-1 gene—is expressed by articular chondrocytes and up regulated in osteoarthritis", ANNALS OF THE RHEUMATIC DISEASES Volume: 65 Published: JUL 2006 Page: 965-967. * |
| 楊寧正，PEDF基因治療於骨關節炎成效分析研究成果報告，行政院國家科學委員會專題研究計畫，民國96年12月10日 * |
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| TW201412327A (en) | 2014-04-01 |
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